Soluble variants of type I membrane proteins, and methods of using them

ABSTRACT

The present invention describes novel soluble variants of type I membrane protein GA733-2 and methods of making and using them. In addition, the present invention describes a method of converting type I membrane proteins into secretory proteins which may be used for active immunotherapy against carcinomas and as reagents in the detection of GA733-2 expression on tumor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/413,805, filed Mar. 30, 1995, which is a divisional of U.S.patent application Ser. No. 08/148,216, filed Nov. 5, 1993, nowabandoned, which is a continuation of U.S. patent application No.07/778,232, filed Oct. 18, 1991, now abandoned.

[0002] This work was performed under grants CA 10815 and CA21124-1 fromthe National Institutes of Health.

BACKGROUND OF THE INVENTION

[0003] This invention relates to novel soluble variants of the type Imembrane proteins and to methods for making and using them. Morespecifically this invention relates to membrane proteins, and to methodsof making and using them in the diagnosis and treatment of cancer.

[0004] The GA733-2 antigen has been found to be associated with avariety of human carcinomas such as colorectal, pancreatic, and breastcarcinoma [D. Herlyn et al, J. Immunol. Meth., 73:157-167 (1984

[0005] ); H G. Gottlinger et al, Int. J. Cancer, 38:47-53 (1986)].PGA733-2 is a 40 kDa human cell surface glycoprotein antigen that isassociated with carcinomas of various origins. Its biological functionremains unknown. Hydrophobicity analysis of the protein sequencepredicted by cDNA has suggested that the GA733-2 antigen is a type Imembrane protein, i.e., it possesses signal peptide, extracellulardomain, trans-membrane domain and intracellular anchor. Anamino-terminal 23 residue signal peptide is followed by a 242 residueextracellular domain containing 12 cysteine residues and 3 potentialN-glycosylation loci, a 23 residue trans-membrane domain, and a highlycharged 26 residue intracellular anchor [S. Szala et al, Proc. Natl.Acad. Sci. (USA), 87:3542-3546 (1990); see also, M. S. Perez and L. E.Walker, Journal of Immunology, 142:3662-67 (1989); J. Strnad et al,Cancer Res, 49:314-17 (1989); and B. Simon et al, Proc. Natl. Acad. Sci.(USA), 87:2755-59 (1990)]. PGA733-2 is a monoclonal antibody (mAb)defined antigen [A. H. Ross et al, Biochem. Biophys. Res. Comm.,135:297-303 (1986)]. Several independently derived mAbs GA733, CO17-1A,M77, M79, 323/A3, among others, all define the GA733-2 antigen [See, forexample, D. Herlyn et al, J. Immuno. Methods, 73:157-176 (1984), M.Herlyn et al, Proc. Natl. Acad. Sci. (USA), 75: 1438-1482 (1979) and M.Herlyn et al, Hybridoma, 5: S3-S10 (1986) for discussion of CO17-1A; H.G. Gottlinger et al, supra, for discussing of M77 and M79; and D. P.Edwards et al, Cancer Res., 48:1306-1317 (1986) for discussion of323/A3].

[0006] Monoclonal antibodies that define tumor cell surface antigens areoften being evaluated for the diagnosis and immunotherapy of cancer.Initial studies of mAbs CO17-lA and GA733 have demonstrated bothcytotoxic effects in vitro and tumoricidal responses in vivo inexperimental animal models. Clinical trials have shown strong mAb tumorbinding [D. Herlyn et al, “Initial Clinical Evaluation of Two MurineMonoclonal Antibodies for Immunotherapy of Gastrointestinal Carcinoma,”Am. J. Clin. Oncol., (in press) (1991)]. Cases of partial and completeregression of disseminated cancer have also been reported [H. F. Searset al, J. Biol. Resp. Mod., 3:138-150 (1984); and J. E. Frodin et al,Hybridoma, 7:309-321 (1988)]. Since only microgram quantities of thenative antigen are available, therapeutic approaches to date have beenlimited to passive immunization with mAb and active immunization withanti-idiotype mAb (Ab2).

[0007] Molecular clones for the GA733-2 antigen have been isolated byimmunoselection of COS cells transfected with a cDNA expression libraryderived from a human colon carcinoma cell line [S. Szala et al, Proc.Natl. Acad Sci. (USA), 87:3542-3546 (1990)]. The GA733-2 sequence isidentical to independently isolated cDNAs encoding theadenocarcinoma-associated antigen [J. Strand et al, Cancer Res.,49:314-317 (1989); and M. E. Perez and L. E. Walker, J. Immun.,142:3662-3667 (1989)] and the epithelial glycoprotein antigen [B. Simonet al, Proc. Natl. Acad. Sci. (USA), 87:2755-2759 (1990)]. The GA733-2coding region is 54% identical to the GA733-1 gene, a retroposon that isabundantly transcribed in pancreatic carcinoma cell lines [A. J.Linnenbach et al, Proc. Natl. Acad. Sci. (USA), 86:27-31 (1989)]. TheGA733-2 chromosomal gene contains exons encoding an epidermal growthfactor-like repeat and a thyroglobulin type I repeat [A. J. Linnenbach,unpublished observation).

[0008] To date, other human tumor-associated antigens have beenexpressed in the vaccinia virus vector system: the epithelial tumorantigen expressed by breast carcinomas [M. Hareuveni et al, Proc. Natl.Acad. Sci. (USA), 87:9498-9502 (1990)]; and the melanoma-associatedglycoprotein p97[C. D. Estin et al, Proc. Natl. Acad Sci. (USA),85:1052-1056 (1988); and C. D. Estin et al, J. Natl. Cancer Inst.,81:445-448 (1989)].

[0009] Recombinant p97 antigen has induced specific humoral, cellular,and protective immunity in mice, and humoral and cellular immunity inmonkeys. These observations emphasize the potential usefulness of arecombinant human tumor-associated antigen as vaccines for cancerpatients.

[0010] Furthermore, the preparative isolation of secretable proteindomains containing a specific region of interest (i.e., epitopes) whichcan be used in biological, immunological, or physical (i.e.,crystallography) assays, implies extended possibilities for studying thenature and function of other membrane proteins.

[0011] The baculovirus-insect cell expression system has been wellrecognized for its ability to abundantly express recombinant proteinswhich most often resemble native protein with respect to function,immunoreactivity, and immunogenicity. Baculovirus has been exploited forproduction of a variety of enzymes, trans-membrane proteins, andsecretory proteins such as tissue plasminogen activator, interleukin-2,and human beta interferon. A soluble variant of the cell surface proteinCD4 has been generated by expressing a restriction enzyme cleavedportion of the CD4 cDNA [R. E. Hussey et al, Nature (Lond.), 331:78-81(1988)].

[0012] There remains a need in the art for an easily obtained andpurified peptide which has the antigenicity of the GA733-2 membraneprotein antigen in order to pursue immunological, physical andbiochemical studies of this membrane protein, and to provide a means fordiagnosis and immunotherapy against cancer.

SUMMARY OF THE INVENTION

[0013] In one aspect the present invention provides a polypeptide,designated GA733-2E. This polypeptide is a encoded by a truncated,modified version of native GA733-2 DNA, and permits secretion of animmunogenic fragment of the native GA733-2 antigen into the culturemedium. This novel GA733-2 E truncated antigen surprisingly retains theimmunoreactivity and immunogenicity of the native full length GA733-2antigen, and advantageously, may be easily purified from the culturemedium into which it is secreted.

[0014] A further aspect of this invention is a pharmaceuticalcomposition comprising GA733-2E as an active ingredient together with atleast one substance selected from conventional pharmaceutical carriers,diluents, excipients and adjuvants. Optionally, GA733-2E may also beadmixed with other active ingredients, including other GA733-2 variantpeptides and other, known, cancer treating compounds. This compositionmay be used to elicit an immune response in a subject in a vaccineformulation. This composition is particularly useful in the treatment ofvarious cancers.

[0015] Another aspect of this invention involves a method of eliciting aprotective immune response in patients to certain tumors bearing aGA733-2 antigen by administering an effective amount of the apolypeptide and/or pharmaceutical composition described above. Suchcarcinomas include, but are not limited to, colorectal, pancreatic andbreast carcinomas.

[0016] In yet another aspect, this invention provides diagnosticreagents, which include either the GA733-2E polypeptide orpolynucleotide sequence, which sequence may be optionally associatedwith a detectable label, or bound to a solid support.

[0017] Thus still a further aspect of the invention are methods fordiagnosing carcinomas which are characterized by the expression ofnative GA733-2 antigen, which methods employ the recombinant GA733-2polypeptide or polynucleotide sequences of the invention. Thus, thepresent invention provides a diagnostic kit for the detection of nativeGA733-2 antigen containing these diagnostic reagents.

[0018] Further, the invention provides the recombinant polypeptideGA733-2E, which may be used as a reagent for diagnostic purposes, and inmethods for purifying and isolating the native antigen GA733-2.

[0019] In still another aspect, the invention provides a method ofmaking soluble variants of GA733-2 proteins by omitting native GA733-2DNA sequences for the trans-membrane and cytoplasmic domains, andcreating a secretory protein from the extracellular domain. Preferably,this is accomplished via polymerase chain reaction (PCR).

[0020] Another aspect includes obtaining a soluble variant of any type Imembrane protein produced by the method of omitting the DNA sequencesfor the trans-membrane and cytoplasmic domains from the DNA sequence ofsaid type I membrane protein. This truncated DNA sequence may then beproduced by culturing a selected host cell transfected with a truncatedDNA sequence in operative association with a regulatory sequence capableof directing the expression of the soluble variant. The soluble variantmay also be produced by conventional synthesis.

[0021] Other aspects and advantages of the present invention aredescribed further in the following detailed description of preferredembodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 illustrates the DNA and amino acid sequences of thetruncated GA733-2E soluble variant of the invention [SEQ ID NO: 1 and2].

[0023]FIG. 2 depicts the construction of baculovirus transfer vectorsfor recombinant GA733-2F and GA733-2E.

[0024]FIG. 3 demonstrates the immunogenicity of secreted recombinantantigen GA733-2E vs. GA733-2 and a control bovine serum albumin (BSA).

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides novel soluble variants of a Type Imembrane protein and methods for their preparation and use. Particularlyexemplified in the following discussion of the invention are novelpolypeptides characterized by the immunogenicity of naturally occurringType I membrane protein, the GA733-2 tumor-associated antigen [see,Szala et al, Biochem., 87:3544 (1990), incorporated herein byreference]. However, it is to be understood that the methods applied toproduce the GA733-2 variant proteins and polypeptides, and compositionsand methods utilizing them, may also be applied to other Type I membraneproteins of particular interest.

[0026] One presently preferred polypeptide of the invention isdesignated GA733-2E. As illustrated in FIG. 1 [SEQ ID NO:1 and 2],GA733-2 E is a truncated protein comprising the amino acids 1 through265 of native GA733-2[Szala et al, cited above]. As described in detailbelow in the examples, GA733-2E was prepared from the GA733-2 antigen,using PCR to delete the sequences for the trans-membrane and cytoplasmicdomains from the native GA733-2 antigen, thereby converting theextracellular domain into a secretory protein.

[0027] It has been demonstrated that this truncated polypeptidemaintains the antigenicity of native GA733-2. This characteristic isparticularly surprising because the recombinant GA733-2E is much smallerthan native GA733-2, lacking the 3′ terminal 189 nucleotides of nativeGA733-2 DNA (encoding 49 amino acids from the carboxy terminal of nativeGA733-2 protein).

[0028] The secreted recombinant antigen preparation described hereinmeets an important pre-clinical criteria for a potential vaccine,namely, the ability to elicit antibodies which can bind to humancolorectal carcinoma cells. Both native GA733-2 and recombinant antigenGA733-2E elicited antibodies in mice which specifically andsignificantly bound to colorectal carcinoma cells at serum dilutions ashigh as 1:204,800.

[0029] It is anticipated that one of skill in the art may modify theGA733-2E polypeptide to enable expression of other soluble and secretedvariants. For example, using the techniques described herein as well asknown mutagenic techniques, one may alter one or more amino acids of theGA733-2E sequence, or to add or delete one or more additional aminoacids from the amino or carboxy termini, while retaining the GA733-2immunogenicity. Additionally, replacement of amino acids, e.g.,conservative amino acid replacements, may be used to alter the sequence.

[0030] Conservative amino acid replacements are those that take placewithin a family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarconservative replacement of an amino acid with a structurally relatedamino acid will not have a major effect on its activity

[0031] Such minor modifications to the polypeptide which permit theretention of the GA733-2 immunogenicity are believed to be encompassedby this invention.

[0032] The present invention also encompasses polynucleotide sequences,preferably DNA sequences, which encode the polypeptides described hereinand sequences complementary thereto. A complementary sequence includes acontiguous sequence of nucleotides capable of selectively hybridizing tonative GA733-2 or a complement thereof The GA733-2E nucleotide sequenceis reported in FIG. 1 [SEQ ID NO: 1], and contains the triplet TAA,encoding a stop codon, inserted immediately after the lysine-encodingcodon at nucleotide number #902[See FIG. 1]. The presence of thisinserted stop codon deletes from the native GA733-2 nucleotide sequenceboth the trans-membrane and cytoplasmic domains, that is, the sequenceencoding the 49 amino acid carboxy terminus of native GA733-2.Advantageously expression of this sequence in a selected host cellsystem allows facile secretion of the immunogenic GA733-2E polypeptideinto the culture medium.

[0033] Polynucleotide sequences of this invention also include modifiedGA733-2E sequences, which modifications may be due to allelic variations(naturally-occurring base changes in the species population which may ormay not result in an amino acid change). Similarly, modified DNAsequences which contain substantially the above described deletion andwhich code for the antigenic portion of native GA733-2 are also includedin this invention. Such modified DNA sequences include those whichdiffer in codon sequence due to the degeneracies of the genetic code, aswell as variations in the modified DNA sequence encoding the antigenicportion of native GA733-2 which are caused by point mutations or byinduced mutations to enhance the activity, half-life or production ofthe peptide encoded thereby. Such modifications may include additionalnucleotide deletions or additions at the 5′ and 3′ termini. Typically,such modified polynucleotide sequences differ by only 1 to about 4 codonchanges.

[0034] Other examples of DNA analogs include DNA sequences containingthe above deletion and encoding a GA733-2E polypeptide with minor aminoacid variations from the natural amino acid sequence of GA733-2; inparticular, conservative amino acid replacements.

[0035] Utilizing the sequence data, it is within the skill of the art toobtain other DNA sequences encoding GA733-2E. For example, the gene maybe manipulated by varying individual nucleotides, while retaining thecorrect amino acid(s), or varying the nucleotides, so as to modify theamino acids, without loss of antigenicity.

[0036] The polypeptides of the present invention are prepared preferablythrough use of conventional recombinant, genetic engineering techniquesknown to one of skill in the art. See, e.g., Sambrook et al, “MolecularCloning. A Laboratory Manual.”, 2d edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989). Similarly useful inpreparing polypeptides of this invention is the polymerase chainreaction (PCR) technique and known modifications thereof [See, e.g.,Saiki et al, Science, 239:487 (1988)]. It is also possible to producepolypeptides of this invention by resort to chemical synthesis ofoverlapping polymers. These and other techniques described below arewithin the skill of the art.

[0037] The polypeptides of the present invention may be obtained byapplying PCR technology to native GA733-2, a type I membrane protein, toobtain a secretable variant of that protein. As described in theexamples below, the positioning of the 3′ oligonucleotide primer for thePCR synthesis of the extracellular domain of the GA733-2 antigen isdetermined by hydrophobicity analysis, which located a 23 amino acidhydrophobic domain flanked by positively charged residues. This regionwas interpreted as representing the transmembrane domain. Once thetruncated GA733-2E antigen is prepared by PCR, it may be incorporatedinto a selected vector for expression in a recombinant expression systemas described below.

[0038] One method for producing the polypeptide of the inventionrecombinantly, involves introducing the truncated polypeptide, e.g.GA733-2E, into an eukaryotic expression vector to make an expressionsystem for the recombinant polypeptide. Non-eukaryotic expressionsystems are not desirable because proper folding is not obtained. Aselected host cell is transformed with the vector and cultured. Themethod of this invention therefore comprises culturing a suitableeukaryotic cell or cell line, which has been transformed with a DNAsequence coding for expression of a polypeptide characterized by theimmunogenicity of native, purified GA733-2 under the control of knownregulatory sequences.

[0039] Suitable host cells or cell lines for expression of therecombinant polypeptides of this invention may include insect cells,mammalian cells, yeast cells and other fungal cells. The selection ofsuitable mammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. See e.g., Gething and Sambrook, Nature, 293:620-625(1981), or alternatively, Kaufman et al, Mol. Cell. Biol.,5(5):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446. Normaldiploid cells, cell strains derived from in vitro culture of primarytissue, as well as primary explants, are suitable. Candidate cells maybe genotypically deficient in the selection gene, or may contain adominantly acting selection gene. Suitable mammalian cell lines include,but are not limited to, Chinese Hamster ovary cells (CHO), HeLa, mouseL-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK orHaK hamster cell lines, monkey COS-1 cell lines and the CV-1 cell line.

[0040] Many strains of yeast and other fungal cells known to thoseskilled in the art are also available as host cells for expression ofthe polypeptides of the present invention. However, insect cells arecurrently the preferred host cells in the method of the invention. Seee.g., Miller et al, Genetic Engineering, 8:277-298 (enum Press 1986) andreferences cited therein. Suitable insect expression systems, of boththe lytic and non-lytic type, are well known to those of skill in theart. For example, baculovirus cell expression systems are commerciallyavailable from sources such as Invitrogen [San Diego, Calif.].Techniques for expression using such systems are also known to thoseskilled in the art. [See, e.g., Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987)]. Further, it iscontemplated that the use of a non-lytic baculovirus system, such asthat described by D. Jarvis et al, J. Biotech., 8:1950 (1990), may beadvantageous in producing the recombinant polypeptide of the invention.

[0041] The present invention also provides recombinant molecules orvectors for use in the method of expression of the novel GA733-2Epolypeptide and other polypeptides of this invention. These vectorscontain the DNA sequence, e.g., the GA733-2E DNA sequence, illustratedin FIG. 1 [SEQ ID NO:1 and 2], which alone or in combination with othersequences, encodes an immunogenic fragment of GA733-2.

[0042] The vector employed in the method also contains selectedregulatory sequences in operative association with the DNA codingsequences of the invention. These regulatory sequences are capable ofdirecting the replication and expression of the DNA coding sequences inselected host cells. Regulatory sequences include promoter fragments,terminator fragments and other suitable sequences which direct theexpression of the protein in an appropriate host cell. One of skill inthe art may select from among known regulatory sequences depending uponthe selected host cell to be used in expressing the polypeptides of thisinvention.

[0043] Appropriate expression vectors of which numerous types are knownin the art for mammalian, fungal, and preferably insect, expression canalso be selected by one of skill in the art for this purpose.

[0044] Once expressed in a selected host cell system, the resultingpolypeptide is desirably secreted into the cell culture. Because thepolypeptides of this invention share immunoreactivity and immunogenicitywith the native antigen, antibodies to the native antigen may beutilized to isolate the recombinant GA733-2E from the culture medium byconventional methods. The examples below provide a purification schemefor isolating the soluble protein from the culture medium.

[0045] In an embodiment of this invention, GA733-2E DNA was cloned intothe baculovirus transfer vector pVL 1392[Invitrogen Corp., San Diego,Calif.] and introduced into Autographa californica nuclear polyhidrosisvirus by homologous recombination. Spodoptera frugiperda (Sf9) cellsinfected with the GA733-2E DNA baculovirus recombinant abundantlysecreted a 31 kDa glycoprotein into the culture medium. Recombinantsecretory antigen displayed an in vitro immunoreactivity to mAb and anin vivo immunogenicity in mice that were similar to native antigen.

[0046] The GA733-2E recombinant antigen produced by Sf9 insect cellsresembles the native antigen produced by human cells in severalrespects. The GA733-2E recombinant antigen undergoes N-linkedglycosylation and is immunoreactive to anti-GA733-2 monoclonalantibodies (mAb) in vitro. As determined in either immunoblotting orenzyme linked immunosorbent assay (ELISA), the recombinant antigenreacts with a group of four mAbs defining three different epitopes(GA733, CO17-1A; and M77/M79).

[0047] The applicability of this approach, i.e., modification via PCR toremove the trans-membrane and cytoplasmic domains, expression in aselected host cell system and purification from culture supernatants, toother type I membrane proteins will be a function of the accuracy oflocating their trans-membrane domains. One advantage of this system inthe production of recombinant soluble Type I membrane protein variantsis that detergent-free, recombinant antigen can be purified fromserum-free culture media by mAb affinity column chromatography,preferably in one purification step. Thus, a recombinant protein orpolypeptide characterized by the immunogenicity of the native antigencan be obtained without the necessity of extensive culturing of humancarcinoma cell lines and laborious extraction procedures.

[0048] The method for recombinant expression described herein may alsobe employed to express recombinant GA733-2, which recombinant protein isreferred to in the examples below as GA733-2F. Recombinant GA733-2Fwhich is characterized by the entire amino acid sequence of the nativeantigen is expressed within the cell. Therefore isolation of thisrecombinant antigen differs from that of GA733-2E in that the fulllength recombinant antigen is not secreted from the cell and must beobtained from the cell lysate by conventional means. However, in allother respects, the presently described method of production of GA733-2Emay be employed to obtain recombinant GA733-2F.

[0049] The polypeptides described herein and produced by the methodsdescribed above may be used in therapeutic, vaccinal and pharmaceuticalcompositions to elicit an active immune response in a subject. Forexample, the protective effects of injections with the GA733-2E antigenmay be evaluated using an animal tumor model. Thus, the antigens of thisinvention are desirable for use in vaccine compositions.

[0050] These polypeptides are particularly useful in the treatment andimmunization for various cancers. The active immunotherapy of cancerwith purified tumor-associated antigens requires that antigen DNA bemolecularly cloned and expressed, since these antigens cannot bepurified in sufficient quantities from cultured human tumor cells.

[0051] The therapeutic mechanism of the compositions and methods of thepresent invention differs in principle from that of the large majorityof drugs for treatment of carcinoma in use at the present time. Theimmunogens of the present invention display highly specific activity ingenerating active immunity against the tumors, so that patients do notsuffer the many disadvantages of conventional cancer therapy.Alternatively, the recombinant antigen of the invention may be used incombination with known tumoricidal agents. The tumors susceptible totreatment or immunization by the present method and compositionsinclude, but are not limited to, colorectal, pancreatic, and breastcarcinomas. Other tumor types which express GA733-2 can be identified byvarious methods known in the art.

[0052] The pharmaceutical and vaccinal compositions of the inventioncomprise a therapeutically effective amount of a polypeptide of thisinvention in admixture with a pharmaceutically acceptable carrier. Thepharmaceutical compositions may be utilized in conventional typeformulations such as, particularly, injectables.

[0053] In one embodiment of this invention, the therapeutic andpharmaceutical compositions of the invention may include GA733-2Epolypeptide in admixture with an adjuvant. Suitable adjuvants are wellknown to those of skill in the art and include saponins such as Quil A,aluminum hydroxide, lipid A (particularly when incorporated into aliposome carrier), bacillus calmette guerin (BCG), glucan, andcombination products, such as DETOX [Ribi Immunochemical Research, Inc.,Hamilton, Mont.]. DETOX contains detoxified endotoxin (monophosphoryllipid A), cell wall skeletons of Mycobacterium, and squalene. Thisproduct has recently been reported to be particularly useful for use incancer patients. [Mitchell et al, Cancer Res., 48:5883 (1988)].Optionally, these compositions may also include other modified GA733-2polypeptides.

[0054] It is further contemplated that pharmaceutical and vaccinalcompositions containing GA733-2E as active immunogen may also containimmunomodulators such as cytokines, including interleukins andinterferons Suitable immunomodulatory dosages can be readily determinedby one of skill in the art. When these agents are combined in apharmaceutical composition with the type I membrane antigen variants ofthis invention, it is anticipated that each active ingredient will bepresent in the combined composition in the same concentration orslightly lower concentration than if the active ingredient wasadministered alone.

[0055] Suitable carriers are well known to those of skill in the art ofpharmacology [see, e.g., Remingtons Practice of Pharmacy, 9th, 10th and11th Ed.]. To a large extent, selection of the carrier depends upon theadjuvant selected. For example, if aluminum hydroxide or glucan isselected as the adjuvant, sterile saline would be an appropriatecarrier, if BCG is the adjuvant, an oil would be an appropriate carrier.Therefore, exemplary carriers may include sterile saline, lactose,sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil,olive oil, sesame oil, squalene and water. The pharmaceuticalcompositions of the invention may also be administered via liposomesaccording to known techniques. In addition, dependent upon the adjuvantselected, e.g. BCG, the carrier or diluent may include a time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax.

[0056] The formulations of the pharmaceutical composition containing thepolypeptides of this invention may conveniently be presented in unitdosage form and may be prepared by any of the conventional methods.Alternatively, the composition may be in a form adapted for slow releasein vivo, as is known in the art. All methods include the step ofbringing into association the active ingredient with the carrier whichmay constitute one or more accessory ingredients.

[0057] The pharmaceutical and vaccinal compositions of this invention,e.g., containing the GA733-2E as the active material, may be used as aimmunotherapeutic. These compositions may be administered parenterally,or by other appropriate routes. The parenteral route is presentlypreferred and compositions may be prepared by dissolving the compound ina suitable solvent such as an aqueous buffer and dimethyl sulfoxide orglycerol. The parenteral route may be intramuscular, intravenous,intradermal, or subcutaneous.

[0058] The concentration of the novel polypeptides, e.g., therecombinant, modified GA733-2 antigens, in combination with apharmaceutical carrier can be determined by the attending physician,taking into consideration various factors. These factors, which maymodify the action of drugs, include e.g. the condition, body weight, sexand diet of the patient, the severity of the tumor, time ofadministration and other clinical factors. The dosage of thecompositions of the invention used to treat or immunize against thespecific disease condition described herein may be varied depending onthe particular disease and the stage of the disease. Generally, thedaily regimen should be in the range of about 1 μg to about 1000 mg ofGA733-2E polypeptide, or analogs thereof, and preferably 0.01 to about100 mg per kg body weight of a subject. A desirable dosage regimen mayinvolve administration of one to three doses of the vaccine composition,where the antigenic content of the composition is as stated above.

[0059] In addition to vaccinal or therapeutic use for the humandisorders described herein, the methods and compositions of thisinvention may be utilized for veterinary purposes in the treatment of,or immunization of an animal against, carcinomas that afflict othermammals, including horses, swine and cattle, and fowl, for example.These disorders may be treated using similar vaccinal and therapeuticquantities of the polypeptides of this invention that may be used intreating the disorders described hereinabove.

[0060] The polypeptides of the present invention may also be used fordiagnostic, as well as therapeutic, purposes. According to the method ofthe present invention, where desired, primary tumor tissue from apatient can be assayed for GA733-2 expression prior to treatment using apolypeptide of this invention. For example, antibodies to native GA733-2antigen which are present in patient serum may be detected in vitro byreaction with a polypeptide of the present invention. In situhybridization may also be an effective method of diagnosis. Other meansof ascertaining tumor expression of GA733-2 surface antigen are knownand may be used in order to ascertain the probable effectiveness ofimmunotherapy with the polypeptides described herein.

[0061] A polypeptide of this invention, e.g., GA733-2E, may be used in acompetitive immunoassay with labelled antibodies to GA733-2. One suchcompetitive assay involves the use of unlabelled GA733-2E polypeptideswhich are bound to a solid surface, such as latex particles or adipstick. A labelled antibody is incubated with either a standardantigen or a tissue sample and is then put in contact with the solidsurface. The GA733-2E antigen competes with the native GA733-2 antigenfor binding to the labelled antibody. In the absence of any nativeantigen in the tissue sample, a visual label appears. Expression ofGA733-2 on the tumor cell surface would be indicated by the absence of adetectable label.

[0062] In another known system, the monoclonal antibody is bound to asolid surface. The solid phase is then incubated with a standard, e.g.,unlabelled GA733-2E polypeptide, or with a test sample. LabelledGA733-2E polypeptide is then placed in contact with solid surface. Theabsence of a detectable label indicates that the sample expressedGA733-2 antigen.

[0063] In yet another aspect, the polypeptide of the invention, ormonoclonal antibodies directed thereto, could be adapted for use incommercially available assay systems, such as the double determinanttest kits which are commercially available. Briefly, this involves theuse of two monoclonal antibodies, each directed to a different antigenicdeterminant. A MAb to a first antigenic determinant is bound to thesolid surface, which is then incubated with either standard purifiedantigen, e.g., the GA733-2E polypeptide of this invention, or a tissueextract sample. A labelled MAb to a second antigenic determinant is thenadded to the plate.

[0064] When the GA733-2E polypeptide is used as a reagent in competitiveimmunoassay, detectable labels for attachment to the polypeptide may beeasily selected by one skilled in the art of diagnostic assays. Labelsdetectable visually are often preferred for use in diagnostic kits andin clinical applications due to the rapidity of the signal and its easyreadability. For colorimetric detection, a variety of enzyme systemshave been described in the art which will operate appropriately in thehomogenous assay. As one example of enzyme 1, glucose oxidase, whichuses glucose as a substrate, may be employed. Interaction betweenglucose and glucose oxidase releases peroxide as a product. Enzyme 2 maytherefore be peroxidase, which reacts with peroxide and a hydrogen donorsuch as tetramethyl benzidine (TMB) producing an oxidized TMB that isseen as a blue color.

[0065] In the assays employing colorimetric enzyme systems, such ashorseradish peroxidase (HRP) or alkaline phosphatase (AP), the reactionshould be read within approximately 5 to 15 minutes, preferably 10minutes, to obtain an accurate result. A longer reaction time can leadto color changes induced by trace amounts of enzyme remaining on thereaction surface. Where desirable, a “stop” solution can be employed todisable the enzyme from further reaction after the above 5 to 15 minuteperiod. It is known, for example, that sulfuric acid may be added tostop the reaction of HRP.

[0066] Other such proximal enzyme systems are known to those of skill inthe art, including hexokinase in conjunction with glucose-6-phosphatedehydrogenase which reacts with ATP, glucose, and NAD+ to yield, amongother products, NADH that is detected as increased absorbance at 340 nmwavelength. The loss of absorbance at 340 nm wavelength by the oxidationof NADH is another indicator of positive results in either allostericactivation using phosphofructokinase in conjunction with phosphoenolpyruvate carboxylase and substrates fructose-6-phosphate and NADH orallosteric inhibition using aspartate aminotransferase in conjunctionwith phosphoenol pyruvate carboxylase and substrates oxalacetate,glutamate and NADH. Also, bioluminescence or chemiluminescence can bedetected using, respectively, NAD oxidoreductase with luciferase andsubstrates NADH and FMN or peroxidase with luminol and substrateperoxide.

[0067] Other label systems that may be utilized in the methods of thisinvention are detectable by other means. For example, one desirablemethod employs colored latex microparticles, such as those marketed byBangs Laboratories of Indiana. These latex particles in which isembedded a dye may be used in place of enzymes to form conjugates withthe antibodies and recombinant GA733-2E of the invention and provide avisual signal indicative of the presence of the resultingantibody-antigen complex in applicable assays. The use of such particleseliminates the need for subsequent substrate addition steps, and avoidsenzyme and substrate reagent instability.

[0068] Other conventional label systems that may be employed includefluorescent compounds, radioactive compounds or elements, orimmunoelectrodes. These and other appropriate label systems are known tothose of skill in the art. The selection of a detectable label or labelsystem is merely conventional. Similarly a wide variety of solidsupports are available to the art, such as latex beads. Selection ofsolid supports and the association thereof with polypeptides of thisinvention are also considered conventional.

[0069] The methods and polypeptides described herein may be efficientlyutilized in the assembly of a diagnostic kit. Such a diagnostic kitcontains the components necessary to practice one or more of the assaysdescribed above (or other conventional assay systems) for the expressionof GA733-2 surface antigen on tumor cells Thus, for homogeneous assaysthe kit may contain a monoclonal antibody directed to a native GA733-2,which Mab is associated with a first enzyme, a vial for containing theurine sample, and the synthetic GA733-2E polypeptide conjugated to thesecond enzyme, which in proximity to the first enzyme, produces avisible product. Other conventional components of such diagnostic kitsmay also be included.

[0070] Alternatively, a kit may contain a monoclonal antibody specificfor GA733-2 antigen bound to a solid surface and associated with a firstenzyme, the synthetic GA733-2E polypeptide associated with a secondenzyme, and a sufficient amount of the substrate for the first enzyme,which, when added to the tumor sample and reagents, provides thereactant for the second enzyme, resulting in the color change in theabsence of native GA733-2.

[0071] For an agglutination assay kit, the latex beads would be bound tothe monoclonal antibodies generated against synthetic GA733-2Epolypeptide.

[0072] Where the detectable label present in association with theantibody is designed for non-visual detection, e.g., forradioimmunoassay, the standard components necessary for this assay,e.g., controls, standards and the like, are included in the kit. In thediagnostic kits of this invention, reagents are included which define aclear cut end to the color development step, such as the stop solutionsdescribed above

[0073] In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only, andshould not be construed as limiting this invention in any way.

EXAMPLE 1 PCR Synthesis of GA733-2 Extracellular Domain

[0074] Oligonucleotide primers were designed for PCR synthesis of atruncated cDNA that encodes the amino-terminal signal peptide sequenceand the extracellular 242 amino acids of the selected antigen,GA733-2[R. K. Saiki et al, “Enzymatic Amplification of β-globin GenomicSequences and Restriction Site Analysis for Diagnosis of Sickle CellAnemia,” Science, 230: 1350-1354 (1985)].

[0075] Oligonucleotide primers were synthesized by automatedphosphoramidite chemistry on a model 380 A DNA synthesizer [AppliedBiosystems]. PCR synthesis was performed with GA733-2 cDNA template (1ng) [S. Szala et al, cited above], oligonucleotide primers (1 μM each),and Thermus aquaticus polymerase in a volume of 100 μl.

[0076] The oligonucleotide primers are as follows:

[0077] Primer 1[SEQ ID NO: 3], which corresponds to nucleotides 85-114of native GA733-2 DNA (sense strand)

5′-CCTCTTCTCGGCCTGCAGGCAGCATGGCGC-3′

[0078] and Primer 2[SEQ ID NO:4], which corresponds to nucleotides885-919 of native GA 733-2 DNA (antisense strand)

5′-AGTTACGTCCCAGATTTTATTAGATCTTAACGACA-3′.

[0079] Primer 1 contains a Pst I restriction enzyme site (CTGCAG), whichwas placed II bp upstream of the inherent initiation codon (ATG). Thepositioning of primer 1 considerably reduces the GC-rich 5′ untranslatedsequences, which are thought to impair the translational level of therecombinant product [M. D. Summers et al, “A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experiment Station Bulletin, No. 1555 (1988)]. At the 3′end, Primer 2 introduced a termination codon, TAA (ATT in the antisensestrand), in place of the first amino acid of the putative hydrophobictrans-membrane domain [S. Szala et al]. Additionally, Primer 2 placed anXba I restriction site (AGATCT) immediately adjacent to the stop codon.

[0080] PCR was carried out for 35 cycles (denaturing at 94° C. for 1minute, annealing at 40° C. for 2 minutes, elongation at 72° C. for 3minutes) by using the DNA Thermal Cycler [Perkin Elmer Cetus, Norwalk,Conn. ]. The final elongation step was extended by 7 minutes. Thereaction was extracted with phenol/chloroform and the DNA was purifiedby glass bead adsorption [Bio 101, Inc., La Jolla, Calif. ]. PCRproducts were analyzed in ethidium bromide-stained agarose gels (2%Nusieve, 1% Seakem) [FMC Bio Products, Rockland, Me. ] and the expected˜800 bp cDNA band was visualized.

[0081] This PCR-synthesized, truncated version of the cDNA is termedGA733-2E [SEQ ID NO:1 and 2].

EXAMPLE 2 Preparation of GA733-2E Baculovirus Recombinants

[0082] The construction of a baculovirus transfer vector for GA733-2E[SEQ ID NO:1 and 2], is depicted in FIG. 2. In order to compareexpression of our truncated recombinant, we also constructed arecombinant containing the full length version of GA733-2. Thisrecombinant was designated GA733-2F, also shown in FIG. 2. GA733-2F wasderived by digesting the CDM8 recombinant plasmid with Xba at 37° C. for1 hour [S. Szala et al, supra]. The reaction was stopped by the additionof sodium dodecyl sulfate (SDS) to 0.1% and extracted withphenol/chloroform. The GA733-2F full-length cDNA was inserted into theXba I site of the baculovirus transfer vector pVL1392 [Invitrogen Corp.,San Diego, Calif.], downstream of the AcNV polyhedrin promoter. Therestriction enzymes Pst I and Xba I were used to subclone the GA733-2EPCR product into pVL1392. Optimal conditions for the ligation ofGA733-2E to Xba I digested pVL1392 with T4-ligase [Boehringer-Mannheim,F.R.G.] were an insert:vector molar ratio of 3:1 and a totalconcentration of 5′ ends of 0.4 μM (4.36 μg DNA/10 μl).

[0083] The ligated product was transformed into competent DH 5α E. colicells, and insert-containing clones were identified by bacterial colonyhybridization [M. Grunstein et al, “Colony Hybridization; a Method forthe Isolation of Cloned DNAs that Contain a Specific Gene,” PNAS (USA),72:3961-3965 (1975)].

[0084] The GA733-2E PCR product (Example 1) was digested with Pst I andXba I. Directional cloning into pVL1392 was carried out by using aninsert:vector molar ratio of 3:1 and a total concentration of 5′ ends of0.03 μM (390 ng DNA/10 μl). Recombinant pGA733-2E was isolated and theinsert was sequenced to monitor the fidelity of the PCR synthesis.

[0085] Although T. aquaticus polymerase is known to producemisincorporations, DNA sequence analysis of the pVL1392-2E recombinantcontaining the PCR-derived insert indicated 100% identity to the nativeGA733-2 cDNA sequence [R. K. Saiki et al, Science, 239:487-491 (1988)].Since homologous recombination between the recombinant plasmid transfervectors and the baculovirus genome is a very rare event, two rounds ofenrichment for recombinant virus by dot-blot hybridization (anautoradiography of lysed insect cell microcultures dot-blotted tonitrocellulose membranes and probed with ³²P-labelled GA733-2 cDNA)preceded 1-2 rounds of visual screening for occlusion body negativerecombinant viral plaques. For example, in the second dot-blothybridization screening for GA733-2F, well number F10 was found to beplaque pure on first round of visual screening. The GA733-2E recombinant[SEQ ID NO:1 and 2] was purified in the same manner.

EXAMPLE 3 Generation and Purification of Baculovirus Recombinants

[0086] Sf9 Spodoptera frugiperda insect cells [Invitrogen, Corp., SanDiego, Cailf.] were cultured at 27° C. in supplemented Grace's InsectMedium [Gibco, Gaithersburg, Md.] containing 10% fetal bovine serum(FBS) and antibiotics. An aliquot of 1.5×10⁶ cells was co-transfectedwith 2 μg of CsCl purified pGA733-2F or pGA733-2E, respectively, and 1μg of purified AcNPV viral DNA, by using the calcium chlorideprecipitation method [M. D. Summers et al, supra]. After 7 days ofincubation, a viral stock containing wild-type and recombinant viruseswas harvested.

[0087] Recombinants were enriched by two rounds of dot-blothybridization screening [M.-C. Fung et al, J. Virol. Meth., 19:33-42(1988)]. Fifty μl of serially diluted viral supernatant ( 10⁻²10⁻⁸,first round, 10³¹ ³10⁻⁷, second round) were used to infect 1.5×10⁴ cellsin each well of a 96-well plate. Supernatants were collected 6 days postinfection (p.i.). Cells were lysed in 0.2 N NaOH, vacuum blotted ontonitrocellulose filters [Schleicher & Schuell, Keene, N.H.], andhybridized to a nick translated, ³²P-labelled GA733-2 cDNA insert probe(1×10⁶ counts/mi). Supernatants containing recombinant virus wereindicated by autoradiography of the filters.

[0088] Plaque purification was carried out by 1-2 rounds of visualscreening of infected Sf9 cell monolayers with 0.75% agarose overlay[Seakem, FMC Bio Products, Rockland, Me]. Occlusion body negativeplaques, representing pure recombinant viral colonies were picked 7 daysp.i. Agarose plugs were eluted in culture medium that was used to infect2×10⁶ Sf9 cell monolayers in 25 ml culture flasks, yielding 5 ml of purehigh-titer viral stock supernatants (2-5×10⁸ plaque-forming unit/ml).

EXAMPLE 4 Expression of Recombinant Antigens

[0089] Aliquots of 2×10⁶ Sf9 cells in mid-logarithmic growth phase (99%viable) were infected with the GA733-2E viral recombinant including awild type AcNPV virus control obtained from Invitrogen (Maxbac™) at amultiplicity of infection of at least 10 In the baculovirus life cycle,protein production regularly reaches a maximum 48 to 72 hours p.i.Recombinant protein was therefore harvested 64 hours p.i. The first 24hours of incubation proceeded in TNM-FH medium, i.e., supplementedGrace's insect medium, obtained from Invitrogen. The cells were thencultured for 40 hours under serum-free conditions (Sf900 medium) [Gibco,Gaithersburg, Md. ]. Supernatants were collected and cell pellets wereextracted with a buffer containing nonidet P-40 (0.5% NP-40, 0.14 MNaCl, 0.01 M NaF, 0.01 M Tris (pH 7.5), 5 mM EDTA, 1 mMphenylmethylsufonyl-fluoride and 8 μg/ml aprotinin). Sf9 cell lysateswere clarified by centrifugation at 100,000×g. Cell lysates wereprepared from positive control, colorectal carcinoma SW948 cells [ATCCCCL 237] in the same manner.

[0090] The purification procedure was conducted as follows: (1) BSA andGA733 Ab columns were equilibrated with 20 mM Tris (Sigma T-1503)buffer, pH 7.5 (“wash buffer”); (2) the filtered solution was passedthrough the BSA column; (3) the BSA column flow-through was passedthrough the GA-733Ab column; (4) the column was washed with wash bufferand the wash was collected; (5) the antigen was eluted from the columnwith 50 mM diethyl amine buffer, pH11 (“elution buffer”), and each ofthe 101 ml fractions was neutralized with 1 M TRIS, pH 6(“neutralization buffer”); (6) the column was washed again with 20-30mls wash buffer and the wash was collected.

[0091] We performed a Western blot using the wash from step (1), theflow-through from step (2), the wash from step (4), the eluate from step(5), and the wash from step (6) We then pooled the tubes containing theantigen according to the results of the Western blot. We dialyzed theantigen eluate against PBS, using 3500 MW cutoff dialysis tubing anddetermined protein concentration by Lowry protein assay. The antigen wasthen ready for testing in radioimmunoassay or ELISA.

[0092] Fifty μg of protein (Lowry) from supernatants or detergentextracts were boiled for 90 seconds in non-reducing loading buffer [2%SDS, 62.5 mM Tris-Cl (pH 6.8), 10% glycerol, 0.001% bromphenol blue]prior to electrophoresis in a 15% SDS-polyacrylamide gel [U. K. Laemmli,Nature (Lond.), 227:680-685 (1970)]. Samples were electro-blotted ontonitrocellulose filters (BioRad, Richmond, Cailf.) and reacted with themurine mAb GA733 (1 μg/ml) [described in D. Herlyn et al, J. Immuno.Methods, 73:157-167 (1984)]. Antibody binding to the recombinantproteins was visualized through an alkaline phosphatase conjugatedanti-mouse immunoglobulin G (IgG) [Promega, Madison, Wis.].

[0093] Sf9 insect cells infected with GA733-2F and GA733-2E purifiedrecombinant virus were found by immunoblot analysis to be capable ofexpressing recombinant antigens carrying the epitope defined by mAbGA733. Additional immunoblot experiments established that therecombinant antigen has a second epitope defined by mAb M77 and M79[H.G. Gottlinger et al, Int. J. Cancer, 38:47-53 (1986)]. Reactivity withmAb 323/A3[D. P. Edwards et al, Cancer Res, 46:1306-1317 (1986)] wasalso observed.

[0094] Detergent extracts of cells infected with GA733-2F baculovirusexhibited antigen that appeared heterogeneous in size (34, 37, 40, 44and 76 kDa), with the major 40 kDa species co-migrating with the nativeglycoprotein expressed by SW948 colorectal carcinoma cells. Sf9 controlcells infected with wild-type AcNPV produced no protein bound by mAbGA733. The high Mr species (76 kDa) seen in GA733-2F infected cells mayrepresent an antigen dimer. The antigen expression level of GA733-2F wasnot significantly affected by its 5′ GC-rich untranslated sequence, incomparison to GA733-2E which lacks these sequences. In comparison tocell lysates, the supernatants of GA733-2F infected cells showedrecombinant 40 kDa protein in low amount, probably resulting from cellshearing and cell death during the course of infection, liberatingprotein from the membranes to the supernatant medium.

[0095] As was seen with the GA733-2F antigen, multiple species of theGA733-2E antigen [SEQ ID NO:1 and 2] are also apparent. The detergentextract of cells infected with GA733-2E baculovirus has minor (29, 35kDa) and a major (31 kDa) monomeric species, and possible dimer (62 kDa)and tetramer (112 kDa) species. As anticipated, the GA733-2E baculovirusinfected cells exhibited a greater amount of antigen in the culturesupernatant, compared to recombinants containing the full length versionof GA733-2 (GA733-2F). The GA733-2E antigen [SEQ ID NO:1 and 2] found inthe supernatant appears less heterogeneous, with the 31 kDa speciespredominating. Thus, by removing sequences for the transmembrane andcytoplasmic domains, we converted a type I membrane protein into asecreted protein.

EXAMPLE 5 N-Glycosylation of Recombinant Antigens

[0096] Antigen heterogeneity was studied by culturing infected Sf9 cellsin the presence of two different concentrations of tunicamycin. Wecarried out this N-glycosylation inhibition study for the recombinantGA733-2E antigen [SEQ ID NO: 1 and 2]. Cells were seeded and infected asdescribed above. After 24 hours of incubation, TNM-FH medium wasreplaced by serum-free Sf900 medium containing 2 μg/ml or 5 μg/ml of theN-glycosylation inhibitor tunicamycin [R. T. Schwarz et al, TIBS,5.65-67 (1980)]. After a total incubation time of 64 hours, protein washarvested from the supernatant and from lysed cells as describedearlier. Electrophoresis and immunoblotting was carried out as describedabove with forty μg of protein was applied to each lane.

[0097] Immunoblot analysis with mAb GA733 demonstrated a more uniformappearance of both GA733-2E [SEQ ID NO: 1 and 2] and GA733-2Frecombinant antigens. N-linked glycosylation appeared to have beenblocked almost entirely at concentrations of 2 μg/ml of tunicamycin.

[0098] The diversity of subspecies around the major 40 kDa antigenspecies of GA733-2F was reduced to a predominant 34 kDa species. This isin agreement with the antigen's calculated protein molecular mass of34.9 kDa [S. Szala et al, cited above]. Faint additional bands canpossibly be attributed to the minimal residual N-glycosylation permittedby the concentrations of tunicamycin administered to the Sf9 cells. The76 kDa putative antigen dimer was found to be reduced to 63 kDa bytunicamycin.

[0099] The major 31 kDa form of GA733-2E [SEQ ID NO: 1 and 2] wasreduced to a 29 kDa species, which is consistent with a proteinmolecular mass of 29.8 kDa calculated from the truncated cDNA sequence.The putative 62 kDa antigen dimer was reduced to 55 kDa. Hence, thediversity of subspecies of both recombinant antigens found in theimmunoblot analysis are the result of different stages of the insectcell's glycosylation process and do not represent different proteinprimary structures.

EXAMPLE 6 Immunoreactivities of Native and Secreted Recombinant Antigens

[0100] Secretory GA733-2E was used for further immunological studies andcompared with native GA733-2. mAb affinity chromatography ofsupernatants from large-scale Sf9 cell suspension cultures infected withGA733-2E baculovirus yields an average of ˜1.0 mg of secreted antigenper liter. The full-length recombinant GA733-2F antigen [SEQ ID NO:1 and2] was not further analyzed.

[0101] SW1116 colorectal carcinoma cells and WM9 melanoma cells havepreviously been described [H. Koprowski et al, Somat. Cell Genet.,5:957-972 (1979); and M. Herlyn et al, Cancer Invest., 1:215-224(1983)]. Native GA733-2 antigen was obtained from NP-40 lysate of SW1116tumors grown in nude mice. Detergent extracts were purified on mAb GA733immunoaffinity columns as described previously [A. H. Ross et al,Biochem. Biophys. Res. Comm., 135:297-303 (1986)].

[0102] For large-scale production of secreted recombinant antigen, Sf9cells were infected with GA733-2E recombinant baculovirus at amultiplicity of infection of 5. The cells were cultivated at 27° C. inspinner flasks at 2×10⁶ cells/ml in TNM-FH medium for 24 hours, and thenin serum-free Sf900 medium for an additional 40 hours. The infected Sf9cell culture supernatants were harvested by centrifugation and passedover a mAb GA733 immunoaffinity column.

[0103] Female Balb/c mice, 16 weeks old (Harlan Sprague-Dawley,Indianapolis, Ind.), were immunized s.c. 3 times, at 2 week intervals,with 1 to 5 μg of aluminum hydroxide precipitated [D. Herlyn et al,Proc. Natl. Acad. Sci. (USA), 84:8055-8059 (1987)] native GA733-2antigen, secreted recombinant GA733-2E antigen, or bovine serum albumin(BSA). Mouse sera were obtained before and at 24, 38, 50 and 70 daysfollowing the first immunization.

[0104] Binding of mAb CO17-1A and GA733 to purified tumor antigen wasdetermined in ELISA as previously described [M. Wettendorff et al, J.Immunol. Method., 116:105-115 (1989)]. Wells of microtiter plates werecoated with various concentrations (0.2-5 μg/ml) of antigen or BSA.Binding of mAb or normal mouse IgG (0.4-10 μg/ml) was detected byalkaline phosphatase conjugated goat anti-mouse F(ab′)₂ antibody [CappelLabs, Durham, N.C.] and substrate. Optical densities were measured at405 nm in a Titertek ELISA reader [ICN Biomedicals, Inc., Costa Mesa,Calif.].

[0105] Binding of mouse sera to GA733-2 antigen-positive SW1116 cellsand antigen-negative melanoma cells WM9 was determined in mixedhemadsorption assay as previously described [D. Herlyn et al,“Anti-idiotypic Antibodies Bear the Internal Image of a Human TumorAntigen,” Science, 232:100-102 (1986)].

[0106] Adherent tumor cells were incubated with various dilutions ofmouse sera obtained before and after immunization with native GA733-2antigen, recombinant GA733-2E antigen [SEQ ID NO:1 and 2], or BSA.Binding of serum antibodies to the cells was detected by sheep red bloodcells that had been bound with mouse anti-sheep red blood cellantibodies and rabbit anti-mouse IgG antibodies serving as indicatorcells. Statistical significance of immunological data was calculatedwith Student's t-test.

[0107] Referring to FIG. 3, on day 50 of the experiment (i.e., 22 daysafter the final immunization) both the native GA733-2 antigen and therecombinant GA733-2E antigen induced antibodies binding toantigen-positive SWI116 colorectal carcinoma cells were observed. Nobinding to antigen-negative melanoma cells WM9 was observed. Suchantibodies were not induced by immunizations with BSA (FIG. 3) The serumend point dilutions (i.e., the highest dilutions of experimental serashowing significant (P<0 05) binding versus control sera obtained eitherbefore antigen or after BSA immunization) were similar (˜1:204,800) forboth groups immunized with either native GA733-2 antigen (1μg/injection) or recombinant GA733-2E antigen (2.5 μg/injection) (FIG.3) [SEQ ID NO:1 and 2].

[0108] Furthermore, both antigen preparations induced similar (P>0.05)concentrations of anti-colorectal carcinoma cell antibodies at all serumdilutions tested. Concentrations of colorectal carcinoma cell-bindingantibodies induced with native GA733-2 antigen were somewhat dependenton the dose of the administered antigen (1 μg dose<5 μg dose, P<0.05, atserum dilutions of 12.8, 51.2, and 204.8×10⁻³). Hence, as is often thecase with proteins expressed in the baculovirus system, theimmunogenicity of recombinant and native antigens are similar [V. ALuckow et al, Biotechnology, 6:47-55 (1988)].

[0109] Maximal binding reactivities of mAb CO17-1A [M. Herlyn et al,Hybridoma, 5: S3-S10 (1986)] and mAb GA733 to native GA733-2 antigen,recombinant GA733-2E antigen [SEQ ID NO: 1 and 2], or BSA (negativecontrol) are shown in Table 1, below: TABLE 1 Immunoreactivities ofNative and Secreted Recombinant Antigens Binding of mAb to purifiednative (GA733-2) and recombinant (GA733-2E) antigens were determined byELISA. Maximal optical density (at 405 nm) of antibody binding toantigen^(a) Antibody^(b) GA733-2 GA733-2E BSA CO17-1A 0.60 1.21 0.07GA733 0.86 1.30 0.02 Normal mouse IgG 0 0 0

[0110] Both mAb specifically bound to native and recombinant antigenpreparations, as indicated by the absence of binding of either mAb toBSA, or of normal mouse IgG to tumor antigen. mAb binding to either ofthe two GA733-2 antigen preparations was both mAb and antigenconcentration-dependent.

[0111] Recombinant GA733-2E antigen [SEQ ID NO:1 and 2] showedsignificantly higher (P<0.05) binding reactivity than the native GA733-2antigen. This was consistent with 2 different batches of each antigen.This phenomenon may be related to the different methods used in thepreparative isolation of native membrane antigen and secretedrecombinant antigen. Both the immunoblot and ELISA data indicated thatthe processes of antigen truncation and expression in insect cellsyielded a recombinant antigen with epitopes recognized by mAb raisedagainst native antigen.

[0112] While certain embodiments of the invention have been particularlydescribed, it will be apparent to those skilled in the art that manymodifications and variations may be made. Therefore, the presentinvention is not to be construed as limited by any of the particularembodiments shown, rather its scope will be defined only by the claimswhich follow.

1 4 905 base pairs nucleic acid double unknown DNA (genomic) CDS108..902 1 CCCAGGCCTC GCGCTGCCCG GCCGGCTCCT CGTGTCCCAC TCCCGGCGCACGCCCTCCCG 60 CGAGTCCCGG GCCCCTCCCG CGCCCCTCTT CTCGGCGCGC GCGCAGC ATGGCG CCC 116 Met Ala Pro 1 CCG CAG GTC CTC GCG TTC GGG CTT CTG CTT GCCGCG GCG ACG GCG ACT 164 Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala AlaAla Thr Ala Thr 5 10 15 TTT GCC GCA GCT CAG GAA GAA TGT GTC TGT GAA AACTAC AAG CTG GCC 212 Phe Ala Ala Ala Gln Glu Glu Cys Val Cys Glu Asn TyrLys Leu Ala 20 25 30 35 GTA AAC TGC TTT GTG AAT AAT AAT CGT CAA TGC CAGTGT ACT TCA GTT 260 Val Asn Cys Phe Val Asn Asn Asn Arg Gln Cys Gln CysThr Ser Val 40 45 50 GGT GCA CAA AAT ACT GTC ATT TGC TCA AAG CTG GCT GCCAAA TGT TTG 308 Gly Ala Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ala LysCys Leu 55 60 65 GTG ATG AAG GCA GAA ATG AAT GGC TCA AAA CTT GGG AGA AGAGCA AAA 356 Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg Arg AlaLys 70 75 80 CCT GAA GGG GCC CTC CAG AAC AAT GAT GGG CTT TAT GAT CCT GACTGC 404 Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys85 90 95 GAT GAG AGC GGG CTC TTT AAG GCC AAG CAG TGC AAC GGC ACC TCC ACG452 Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr 100105 110 115 TGC TGG TGT GTG AAC ACT GCT GGG GTC AGA AGA ACA GAC AAG GACACT 500 Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr120 125 130 GAA ATA ACC TGC TCT GAG CGA GTG AGA ACC TAC TGG ATC ATC ATTGAA 548 Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu135 140 145 CTA AAA CAC AAA GCA AGA GAA AAA CCT TAT GAT AGT AAA AGT TTGCGG 596 Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys Ser Leu Arg150 155 160 ACT GCA CTT CAG AAG GAG ATC ACA ACG CGT TAT CAA CTG GAT CCAAAA 644 Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu Asp Pro Lys165 170 175 TTT ATC ACG AGT ATT TTG TAT GAG AAT AAT GTT ATC ACT ATT GATCTG 692 Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr Ile Asp Leu180 185 190 195 GTT CAA AAT TCT TCT CAA AAA ACT CAG AAT GAT GTG GAC ATAGCT GAT 740 Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp Ile AlaAsp 200 205 210 GTG GCT TAT TAT TTT GAA AAA GAT GTT AAA GGT GAA TCC TTGTTT CAT 788 Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser Leu PheHis 215 220 225 TCT AAG AAA ATG GAC CTG ACA GTA AAT GGG GAA CAA CTG GATCTG GAT 836 Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu Asp LeuAsp 230 235 240 CCT GGT CAA ACT TTA ATT TAT TAT GTT GAT GAA AAA GCA CCTGAA TTC 884 Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro GluPhe 245 250 255 TCA ATG CAG GGT CTA AAA TAA 905 Ser Met Gln Gly Leu Lys260 265 265 amino acids amino acid linear protein 2 Met Ala Pro Pro GlnVal Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala 1 5 10 15 Thr Ala Thr PheAla Ala Ala Gln Glu Glu Cys Val Cys Glu Asn Tyr 20 25 30 Lys Leu Ala ValAsn Cys Phe Val Asn Asn Asn Arg Gln Cys Gln Cys 35 40 45 Thr Ser Val GlyAla Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ala 50 55 60 Lys Cys Leu ValMet Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg 65 70 75 80 Arg Ala LysPro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95 Pro Asp CysAsp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110 Thr SerThr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125 LysAsp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys 145 150155 160 Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu165 170 175 Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val IleThr 180 185 190 Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn AspVal Asp 195 200 205 Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val LysGly Glu Ser 210 215 220 Leu Phe His Ser Lys Lys Met Asp Leu Thr Val AsnGly Glu Gln Leu 225 230 235 240 Asp Leu Asp Pro Gly Gln Thr Leu Ile TyrTyr Val Asp Glu Lys Ala 245 250 255 Pro Glu Phe Ser Met Gln Gly Leu Lys260 265 30 base pairs nucleic acid unknown unknown DNA (genomic) 3CCTCTTCTCG GCCTGCAGGC AGCATGGCGC 30 35 base pairs nucleic acid unknownunknown DNA (genomic) 4 AGTTACGTCC CAGATTTTAT TAGATCTTAA CGACA 35

What is claimed is:
 1. A method for treating a mammalian subject with acancer comprising the step of administering to said subject apharmaceutical composition comprising an amount of the truncatedpolypeptide GA733-2E SEQ ID NO: 2 effective to induce anantigen-specific humoral immune response in said subject.
 2. The methodaccording to claim 1, wherein said cancer is selected from the groupconsisting of colorectal carcinoma, breast carcinoma and pancreaticcarcinoma.
 3. The method according to claim 1, wherein said compositioncomprises an adjuvant.
 4. The method according to claim 1, wherein saidcomposition comprises an immunomodulator.
 5. The method according toclaim 4, wherein said immunomodulator is selected from the groupconsisting of a cytokine, an interleukin and an interferon.
 6. Themethod according to claim 1, wherein said composition comprises acarrier.
 7. A composition for active immunotherapy of a mammaliansubject with a cancer comprising an amount of the truncated polypeptideGA733-2E SEQ ID NO: 2 effective to induce an antigen-specific humoralimmune response in said subject.
 8. The composition according to claim7, wherein said cancer is selected from the group consisting ofcolorectal carcinoma, breast carcinoma and pancreatic carcinoma.
 9. Thecomposition according to claim 7, further comprising an adjuvant. 10.The composition according to claim 7, further comprising animmunomodulator.
 11. The composition according to claim 10, wherein saidimmunomodulator is selected from the group consisting of a cytokine, aninterleukin and an interferon.
 12. The composition according to claim 7,further comprising a carrier.